Recently, the demand for discovery of safe anticancer drugs from nature has been extensively increased to avoid cancer chemotherapy side effects. Blepharis edulis (Forssk.) pers (Acanthaceae) is a perennial herb growing in Egypt with many traditional and pharmacological activities. The in vitro assay as well as identification of phytoconstituents in plant samples by LC/MS/MS spectrometry has reserve time and efforts for rapid screening and identification of biologically active compounds in plants. Herein, we investigated the cytotoxic activity of different fractions of Blepharis edulis methanolic extract using Sulforhodamine B stain (SRB) against three cancer cell lines; Hepatocellular carcinoma (HepG-2), human colon carcinoma (HCT-116), and human breast adenocarcinoma (MCF-7) cell lines. The results revealed that the n-butanol fraction showed the most potent cytotoxic activity against the three tested cancer cell lines with CC50 values 9.12 ± 0.92, 6.79 ± 0.65 and 4.19 ± 0.51 against MCF-7, HCT-116 and HepG2 cell lines, respectively. Moreover, the high resolution the ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS-MS) metabolomic analysis for the n-butanol fraction as the most active sample has been achieved for the first time. A total of 30 compound of different classes including flavonoids, aromatic aldehydes, aromatic acids and alkaloids, have been identified from Blepharis edulis.